We study transcriptional and translational mechanisms involved in nutrient control of gene expression in the yeast Saccharomyces, focusing on a regulatory system that induces genes encoding amino acid biosynthetic enzymes in response to starvation for amino acids. The transcriptional activator in this pathway, GCN4, is induced at the translational level in starved cells by phosphorylation of initiation factor 2 (eIF2) by the protein kinase (PK) GCN2. Phosphorylation of eIF2 reduces the concentration of the ternary complex (TC) containing eIF2, GTP, and initiator methionyl-tRNA, which transfers tRNAiMet to the 40S ribosome. This impedes general protein synthesis but induces GCN4 translation by a reinitiation mechanism involving small upstream open reading frames (uORFs) in the GCN4 mRNA leader. A reduction in TC levels allows 40S ribosomes scanning the GCN4 mRNA leader after translating uORF1 to bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead. GCN2 is activated in starved cells by binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like region which functions as a sensor of amino acid availability. Activation of GCN2 also requires the GCN1-GCN20 complex that binds to the N-terminal domain (NTD) of GCN2. GCN1 interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We isolated point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1-GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1-GCN20 and a stronger decrease in eIF2 phosphorylation than did mutating one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells. We showed previously that the wild-type GCN2 PK domain is functionally inert when separated from the HisRS domain, but can be activated by replacement of Arg794 with Gly in the PK domain (R794G). We have determined the crystal structures of the PK domain for wild-type and R794G mutant forms both in the apo state and bound to ATP/AMPPNP. These structures suggest that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving R794. This conformational change is associated with intra-domain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation. Our GCN2 PK structures lack autophosphorylation of Thr882 in the activation loop, an event critical for PK function, and the configuration of active site residues suggests how autophosphorylation facilitates a second step in kinase activation. The orientation of alpha helix C in the PK domain is incompatible with catalysis, allowing the invariant Glu643 (Glu91 in cAPK) to interact with Arg834 (the R of the HRD motif in the catalytic loop) rather than forming the classical salt bridge with invariant Lys628 (cAPK Lys72) in the ?3 of the N-lobe. This is likely responsible for the failure of Lys628 to properly orient the ?- and ?- phosphate groups of ATP. By analogy with other ?RD? kinases, autophosphorylation of Thr882 would enable this residue to displace Glu643 from Arg834 and allow Glu643 to form the salt bridge with Lys628. Previously, we showed that the eIF3 complex, eIF1 and eIF5 reside in a multifactor complex (MFC) with the TC and mapped the interactions between these factors in the MFC. The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the TC. We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui- phenotype) and was lethal in cells expressing the eIF5-G31R mutant protein that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui- phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui- phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo. We have established that optimal transcriptional activation of the amino acid biosynthetic gene ARG1 by GCN4 requires the coactivators SAGA, SWI/SNF, Srb Mediator, and RSC, and that GCN4 recruits these coactivators to target genes in living cells. We recently showed that recruitment of SAGA, SWI/SNF and Mediator to ARG1 is independent of the TATA element and preinitiation complex (PIC) formation, whereas efficient recruitment of the general transcription factors requires the TATA box. This suggests that recruitment of the coactivators precedes PIC assembly. Supporting this prediction, kinetic analysis of coactivator binding at ARG1 reveals that recruitment of SAGA, SWI/SNF and Mediator is nearly simultaneous with binding of GCN4 and is followed 5-10 min later by PIC assembly and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators to ARG1, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (GCN5), a non-HAT function of SAGA, and also Mediator function. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. These interdependencies in coactivator recruitment were also observed at the GCN4 target genes ARG4 and SNZ1. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1, but requires SAGA at ARG4 and SNZ1. We found that that Mediator and SAGA not only stimulate recruitment of the TATA binding protein (TBP), but that all four coactivators enhance Pol II recruitment or promoter clearance following TBP binding, and that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes GCN4 from other yeast activators studied thus far.